Novel use of glucocorticoids for the treatment of epithelial microbial infections of a fluid containing organ with a natural exterior orifice in mammals

ABSTRACT

The disclosure is directed to glucocorticoids for the treatment of epithelial microbial infections in fluid-containing organ having a natural exterior orifice in mammals; this new therapeutic use finds particular interest in the treatment of epithelial microbial infections of ear (such as otitis extema), udder (such as mastitis), and uterus (such as endometritis).

This application is a continuation of U.S. patent application Ser. No.17/263,516, filed Jan. 26, 2021, which is the national stage ofInternational Patent Application No. PCT/EP2019/068619, filed Jul. 10,2019, each of which applications are incorporated herein by reference inits entirety, and claim further priority from European PatentApplication No. 18306029.2, filed Jul. 30, 2018.

The present invention relates to non-invasive and antibiotic needlessmethods for treating epithelial microbial infections in mammals.

More specifically, the present invention is directed to glucocorticoidsfor the treatment of epithelial microbial infections in fluid-containingorgan having a natural exterior orifice in mammals; this new therapeuticuse finds particular interest in the treatment of epithelial microbialinfections of ear (such as otitis externa), udder (such as mastitis),and uterus (such as endometritis).

Numerous animals and people are affected each year with otitis externa;i.e., a painful inflammation of the ear, often accompanied with amicrobial infection of the affected tissue. Animals with ear flaps, suchas many breeds of dogs, are especially susceptible. Their covered outerear canals provide a sensitive environment for inflammation, favoringthe local development of microbes to breed. A variety of bacteria,viruses, fungi and mites can be responsible for causing otitis externa.

Most otic disorders are the result of a painful inflammatory response toinfections, allergic reactions or trauma to the ear. They are often ofinflammatory origin with the bacteria taking advantage and overgrowing.The disordered local microbiome is the result of an incorrectinflammatory/anti-inflammatory balance. An otic infection can beassociated with bacterial, fungal or viral infection and determinationof the precise etiology is not practical since the organism is oftendifficult to isolate and cultivate.

External otitis in animal (contrary to human) is the first localization.Then on account of inflammation, tympanic membrane impairment reachesthe middle ear (tympanic bullae). Tympanic membrane is able to heal andthen both otitis can evoluate separately. Otitis externa (external earinfections), otitis media (middle ear infections) and otorrhea (otitismedia with ruptured ear drum causing effusion) are among the mostprevalent otic disorders.

Otitis externa, involving the ear canal portion of the external ear, isa common otological problem occurring mainly during hot, humid weather.In the incipient stage, symptoms include itching and pain in the earcanal, and tenderness when pressure is applied around the externalauditory canal, the ear lobe is pulled or the jaw is moved. In somecases, suppuration can occur in the ear canal and hearing may bedecreased. Over 90% of cases of otitis externa are due to inflammationassociated with bacterial and fungal infections.

Otitis externa is usually treated with topical application of combinedtherapeutic agents demonstrating antimicrobial and/or antifungalactivity as well as anti-inflammatory action. Broad spectrum topicallyeffective antibiotic otic suspensions containing antibacterial agents,for example neomycin sulfate, colistin sulfate, polymyxin B, orcombinations thereof, all broad spectrum in effect, are utilized todestroy causative bacteria. Antimycotic topically acting agents, forexample nystatin and clotrimazole, are employed to destroy underlyingfungal disease. In addition, the antiviral agent acyclovir is sometimesutilized to treat viral otitis externa including herpes zoster.Anti-inflammatory agents including, for example, hydrocortisone,hydrocortisone acetate and dexamethasone sodium phosphate, oftenincluded in the topically acting suspensions identified above, have beenemployed to control the inflammatory process of otitis externa. Mostoften, antimicrobial and anti-inflammatory agents are utilized incombination to treat the causative, triggering disorder, e.g., microbialinfection, as well as the inflammatory process itself. They are alsomost often administered as suspensions in drop form for topicaladministration to the affected ear in order to enhance and provide amore uniform and sustainable delivery to the ear canal.

Example of a commercial product used to treat otitis externa in dog isSurolan®, which combines miconazole (antifungal), prednisolone(anti-inflammatory agent) and polymyxine B (antibiotic).

Another common epithelial microbial infection is mastitis, especially indomestic animals raised for obtaining milk such as a dairy cow and goat.Mastitis is a disease in which the mammary glands in the animals areinfected with Staphylococcus (e.g., Staphylococcus aureus),Streptococcus, Escherichia coli, or another pathogenic bacterium causinginflammation; or a disease related to such a bacterium. Mastitis is avery costly disease for the dairy industry due to its high incidence,and the resulting reduced milk production, reduced milk quality andincreased culling (removal) of animals from dairy herds. Mastitis isthus a very serious disease in domestic animals, and a known therapyfrequently used is the administration by infusion of an antimicrobialdrug directly into the mammary gland of a domestic animal affected withmastitis. Most of the mastitis is cured or alleviated by such aninfusion for treating mastitis. However, due to a lowering ofsusceptibility of a bacterium causing mastitis to existing antimicrobialdrugs for treating mastitis, at present mastitis for which notherapeutic effect is obtained by known drugs has increased.

Another organ with an exterior orifice in domesticated animals such ascattle commonly subject to epithelial microbial infections is theuterus. Metritis and endometritis are common infections of the uterus,where an incorrect inflammatory balance, usually post-calving, allowsthe development of microorganisms resulting in many metritis cases. Theuse of corticoids until now have been poorly effective.

Antimicrobial resistance has become a global burden for whichinappropriate or excessive antimicrobial use is an importantcontributing factor. In this context, WHO developed severalrecommendations among them it encourages new non-antibioticantimicrobials development.

In this context, the Inventors surprisingly found out thatglucocorticoids, in particular hydrocortisone aceponate, that is wellknown for its anti-inflammatory activity, are able to treat epithelialmicrobial infections in fluid containing organ having a natural exteriororifice, such as but not limited to otitis externa in the ear, mastitisin the udder, and endometritis in the uterus, involving colonization ofpathogenic microorganisms combined to an inflammatory state; thissurprising effect is observed even without the administration of anyantimicrobial agent. The administration of hydrocortisone aceponate(HCA) further leads to the restoration of a normal epithelial microbialflora. This observation is all the more surprising as corticosteroidsare known to induce immunosuppressive effects.

The use of anti-inflammatory agent for the treatment of inflammationdisorders of fluid-containing organs having an exterior orifice has beendisclosed. The international application WO2004/082588 describes suchuse of anti-inflammatory agents (especially selective COX-2 inhibitorydrugs), in specific formulations based with an amphipatic oil and amicrocristallin wax, for the treatment of inflammatory disorders of theear and udder. However, the use of anti-inflammatory agent, particularlyof glucocorticoid agent for the treatment of microbial infectionsthemselves has never been disclosed.

As discussed here above, inflammation of fluid containing organs havinga natural exterior orifice may be associated with the development ofmicroorganisms, conducting to an unbalanced microbial flora whichdevelops abnormally.

Without being bound by theory, the inflammation itself may in fact bethe cause of the abnormal microbial development. The epithelialinflammation promotes the bacteria development and a vicious circlemaintains both inflammation and infection. Nowadays, inflammatorydisorders as well as epithelial microbial infections such as otitis aretreated with common treatments having an action on both inflammation andinfection. Such treatments involve a combination of an antibiotic and/oran antifungal agent with an anti-inflammatory agent. They enable apunctual treatment of the infection, before frequent recurrences.

Nevertheless, the Inventors demonstrated that the use of glucocorticoidssuch as hydrocortisone aceponate (HCA) for treating the inflammationoccurring in an epithelial microbial infection of a fluid containingorgan having a natural exterior orifice resulted in the treatment of theinfection itself.

Glucocorticoids, and in particular HCA, therefore present the advantageof enabling the treatment of both the inflammation and the infection,without affecting the natural microbial flora of the fluid-containingorgan, on the contrary to the use of antibiotic and/or antifungal agentswhich do not only affect the microorganisms responsible of the infectionbut also affect the natural microflora. The unexpected effect ofglucocorticoids on the treatment of infection advantageously decreasesor avoids the use of other antimicrobial agent for the treatment of theinfection. Glucocorticoids such as HCA allow a natural balance toreform, meaning the microbiome restabilises without the need to kill thepathogens, and allowing a more normal natural microbiome aftertreatment.

The Inventors also developed new formulations particularly adapted to animproved and efficient delivery of the glucocorticoid to the fluidcontaining organ having a natural exterior orifice. Those preferredformulations according to the invention present the advantage of havingimproved epithelial adhesive properties leading to a uniform and spreadlocal coating of the area to be treated and therefore to provide a widercapacity of the anti-inflammatory agent to treat the infection locally.

The present invention thus relates to at least one glucocorticoid foruse for the treatment and/or the prevention of epithelial microbialinfections of a fluid containing organ having a natural exterior orificein mammals, preferably a non-human mammal.

The terms “treat”, “treating”, “treatment” and “control”, etc., as usedherein, refer to any action providing a benefit to a subject afflictedwith a condition or disease, including improvement in the conditionthrough lessening or suppression of at least one symptom, delay inprogression of the disease, prevention or delay in the onset of thedisease, etc. The treatment of an epithelial microbial infectionincludes a reduction of the clinical symptoms of the epithelialmicrobial infection and/or an inhibition of the microbialovergrowth/multiplication of pathogenic bacteria, yeasts and/or fungi.

The areas aimed to be treated in the context of the present inventionare fluid containing organs having an exterior orifice. Said organscomprise organs having a natural exterior orifice, such as the ears, theudders, the vagina, the uterus, the mouth, the nostrils . . . .

A fluid-containing organ having a natural exterior orifice according tothe invention includes an ear of an animal. For example, in cats ordogs, the natural exterior orifice of the ear is the orifice of theexternal auditory canal.

A fluid-containing organ according to the invention also includes amammary organ, for example an udder of a milk producing animal such as acow, a goat or a sheep. A “milk producing animal” can be a female of anymammalian species but is preferably an animal raised for the purpose ofproviding milk, e.g., a cow, a goat or a sheep, and encompasses suchanimals whether or not they are lactating at the time of theinflammatory and/or infective condition or at the time of treatment. Thenatural exterior orifice of the mammary organ is the orifice of the teatcanal.

A fluid containing organ further includes a vagina or uterus of ananimal. The natural exterior orifice of the uterus is the vagina. Thenatural exterior orifice of the vagina is the vulva.

Other examples of fluid containing organ having a natural exteriororifice include the digestive tract and the respiratory tract.

Other protected cutaneous or external mucous areas of the body of animalmay also be treated in the context of the present invention such as skinfolds, genital area, underarm or underlegs of animals, interdigital area. . . .

Epithelial microbial infections according to the invention encompassinfection-associated inflammatory disorders of the skin, epithelium orexternal mucous of a fluid containing organ comprising a naturalexterior orifice due to pathogenic microorganisms including bacteria,yeasts and/or fungi. The term “infection” or “infected” may be definedherein by an (abnormal) microbial overgrowth/multiplication ofpathogenic bacteria, yeasts and/or fungi.

Preferably, the present invention encompasses a glucocorticoid for usefor the treatment and/or the prevention of an epithelial microbialinfection of a fluid-containing organ having a natural exterior orificeselected from the group consisting of the ears, the udders and theuterus, in a mammal.

Preferably, the present invention encompasses a glucocorticoid for usefor the treatment and/or the prevention of an epithelial microbialinfection of a fluid-containing organ having a natural exterior orificeby topical application or topical administration of said glucocortocoidonto an epithelial area of the fluid-containing organ having a naturalexterior orifice.

According to a preferred embodiment, the present invention relates tohydrocortisone or a salt thereof, preferably hydrocortisone aceponate,for use for the treatment and/or the prevention of an epithelialmicrobial infection of the ears in a non-human mammal; in such, saidhydrocortisone or a salt thereof, preferably hydrocortisone aceptonate,is preferably used by topical administration onto an epithelial area ofthe ears.

Preferably, the present invention encompasses uses to treat subjectssuffering from otitis externa infections including erythematous andceruminous conditions (erythroceruminous otitis externa).

In a specific embodiment, otitis externa infection are non-suppurativeor non purulent otitis and/or do not involve abnormal tympanic membrane,such as ruptured ear drum.

Preferably, epithelial microbial infections of the ear to be treatedaccording to the invention may be caused by bacteria, fungi and/oryeasts; according to a preferred embodiment, said epithelial microbialinfections of the ear to be treated is caused by bacteria, such asGram-positive bacteria e.g. Staphylococcus aureus and Streptococcus spp.and Gram-negative bacteria Escherichia coli. According to anotherembodiment said epithelial microbial infections of the ear to be treatedis caused by fungi and/or yeasts, e.g. Trichophyton spp., Microsporumspp., Malassezia spp. preferably Malassezia pachydermatis, Candida spp.,in particular by Malassezia spp, preferably Malassezia pachydermatis.

In a most preferred embodiment, epithelial microbial infections of theear to be treated according to the invention may be caused byGram-positive bacteria e.g. Staphylococcus aureus and Streptococcus spp.and Gram-negative bacteria Escherichia coli, fungi and/or yeasts, e.g.Malassezia pachydermatis, Candida spp.

The use according to the present invention also allows the treatment ofmastitis in a domestic animal (mammal).

In a preferred embodiment, the use according to the present inventionalso allows the treatment of mild mastitis in a domestic animal(mammal).

In a preferred embodiment, epithelial microbial infections of udder tobe treated according to the invention may be caused by a bacteriaselected from the group but not limited to Staphylococcus spp. (such asS. aureus, S. chromogenes, S. epidermitis, S. hyicus, S. simulans),Escherichia coli, Klebsiella spp., Trueperella pyogenes, Streptococcusspp. (such as S. agalactiae, S. uberis, S. parauberis, S. salivarius, S.sanguinis, S. dysgalactiae), Corynebacterium spp. (such as C. bovis),Enterococcus spp. (such as E. faecium, E. faecalis, E. saccharolyticus),Pseudomonas spp., Mycoplasma spp. (such as M. bovis, M. alkalescens, M.bovigenitalium, M. bovirhinis, M. californicum, M. canadense),Prototheca spp., Candida spp. (such as C. kefyr, C. humicola, C. rugosa,C. inconspicua, C. krusei, C. lusitaniae), Rhodotorula, Trichosporon,Saccharomyces, Pichia, Cryptococcus, Aspergillus, Penicillum, Epicoccum,Phoma and Alternaria.

In a most preferred embodiment, epithelial microbial infections of theudder to be treated according to the invention may be caused byStreptococcus spp., Staphylococcus aureus, Escherichia coli, Klebsiellaspp., Corynebacterium spp., in particular C. bovis, Mycoplasma spp.and/or Pseudomonas spp.

In another embodiment, the use according to the present invention alsoallows the treatment of metritis and/or endometritis in a domesticanimal (mammal).

In a preferred embodiment, epithelial microbial infections of the uterusto be treated according to the invention may be caused by a bacteriaselected in the group but not limited to Escherichia coli, Trueperella(Arcanobacterium) pyogenes, Gram-negative anaerobic bacteria such asPrevotella melaninogenica and Fusobacterium necrophorum.

In another embodiment, the glucocorticoid is for use for the preventionof an epithelial microbial infection of a fluid containing organ havinga natural exterior orifice in a mammal. The term “prevention” usedaccording to the invention includes the prevention or reduction of riskof recurrence of an epithelial microbial infection of a fluid containingorgan having a natural exterior orifice. The prevention of an epithelialmicrobial infection includes prevention or reduction of risk ofrecurrence of the clinical symptoms of the epithelial microbialinfection and/or an inhibition of the microbialovergrowth/multiplication of pathogenic bacteria, yeasts and/or fungi.

More specifically, the present invention is useful for prevention ofotitis externa, such as otitis including erythematous and ceruminousconditions, mastitis, endometritis and/or metritis.

The subject according to the invention is preferably a mammal, morepreferably a non-human mammal, such as a domesticated farm animal (e.g.,cow, horse, pig) or a pet (e.g., dog, cat).

In case the present invention aims to treat infectious otitis externa,the non-human mammal is preferably a companion animal, including but notlimited to a dog or a cat.

Dogs frequently subjects of otitis externa include but are not limitedto dogs having covered outer ear canal and droopy ears (for examplecocker, dachshund . . . ), abundant hairiness in the ear canal (forexample, poodle, bichon . . . ), narrow ear canal (for example Shar-pei,French bulldog . . . ), hypersecretion of cerumen (for example, Belgianshepherd, German shepherd . . . ).

In a specific embodiment wherein the glucocorticoid according to theinvention is for use in the prevention of otitis externa in a dog, theglucocorticoid is HCA and said dog is preferably not an atopic dog.Preferably said dog is a cocker.

In case the present invention aims to treat udder infections (mastitis),the mammal is preferably a non-human lactating mammal. Examples ofnon-human lactating mammals include ruminants, especially bovid such ascow, buffalo, but also goat, sheep, and camel. Preferably, the non humanlactating mammal is a bovid, even more preferably said animal is a cowor a buffalo.

In case the present invention aims to treat uterus and/or vaginainfections (metritis, endometritis), the mammal is preferably a mammalwith a uterus. Examples of such mammals include but are not limited tohumans, ruminants (bovid including e.g. cow, buffalo, but also goat,sheep, and camel) and sows. Preferably, said mammal is a non-humanmammal, even more preferably said animal is a bovid including cows andbuffalos.

In another embodiment, a subject according to the invention is a mammalwith an epithelial microbial infection due to one or more pathogenicmicroorganisms which are resistant to conventional treatments likeantibiotics.

Glucocorticoids (also called steroids, corticosteroids, or cortisoneanalogues) are compounds that are generally used for local application(nasal, cutaneous, ophthalmic, etc.); said glucocorticoid may be amonoester or a diester; preferably the glucocorticoid of the inventionis a diester.

Glucocorticoid according to the invention may be selected in the groupcomprising alclometasone dipropionate, alclometasone propionate,amcinonide, beclomethasone dipropionate, betamethasone acetate,betamethasone dipropionate, betamethasone sodium phosphate,betamethasone benzoate, betamethasone valerate, budesonide, clobetasolpropionate, clobetasol butyrate, clocortolone pivalate, desonide,dexamethasone acetate, dexamethasone nicotinate, dexamethasonepropionate, dexamethasone valerate, dexamethasone sodium phosphate,desoximetasone, diflorasone diacetate, diflucortolone valerate,halobetasol propionate, flumethasone pivalate, fluocinolone acetonide,fluocinonide, flurandrenolide, fluticasone propionate, halcinonide,halobethasone propionate, halomethasone, a hydrocortisone such asbenzodrocortisone (hydrocortisone 17-benzoate), hydrocortamate(hydrocortisone 21-(diethylamino)acetate), hydrocortisone aceponate(hydrocortisone 21-acetate 17α-propionate), hydrocortisone acetate,hydrocortisone bendazac, hydrocortisone buteprate (hydrocortisone17α-butyrate 21-propionate), hydrocortisone butyrate (hydrocortisone17α-butyrate), hydrocortisone 21-butyrate, hydrocortisone cypionate(hydrocortisone cyclopentanepropionate), hydrocortisone hydrogensuccinate, hydrocortisone phosphate, hydrocortisone sodium phosphate,hydrocortisone sodium succinate, hydrocortisone succinate(hydrocortisone hemisuccinate), hydrocortisone tebutate, hydrocortisonevalerate, hydrocortisone xanthogenic acid and hydrocortisone probutate;methylprednisolone acetate, methylprednisolone sodium succinate,methylprednisolone aceponate, mometasone furoate, prednisolone sodiumphosphate, prednisolone acetate, prednisolone valerate, prednicarbate,prednisone, triamcinolone acetate, triamcinolone diacetate,triamcinolone acetonide,

Preferably, the glucocorticoid is hydrocortisone or a salt thereof; morepreferably glucocorticoid is hydrocortisone diester.

Even more preferably, the glucocorticoid according to the invention ishydrocortisone aceponate.

When used for the treatment and/or the prevention of epithelialmicrobial infections of a fluid containing organ having a naturalexterior orifice in mammals, the at least one glucocorticoid ispreferably formulated in a pharmaceutical composition that includes apharmaceutically acceptable liquid or solid carrier or carriers.

According to a specific embodiment, the at least one glucocorticoid isthe only active agent in the pharmaceutical composition; that is to saythat said pharmaceutical composition does not contain any other activeagent selected from antibiotic agent, antifungal agent or antiviralagent.

The present invention thus relates to a pharmaceutical compositioncomprising hydrocortisone or a salt thereof, preferably hydrocortisoneaceponate, and a pharmaceutically acceptable carrier for the treatmentand/or the prevention of an epithelial microbial infection of the ear ina non-human mammal, wherein said composition does not contain anyantibiotic agent, antifungal agent and/or antiviral agent; preferably,said composition is administered by topical application onto anepithelial area of the ears.

According to the present invention, an active agent is a pharmaceuticalor a veterinary compound showing a therapeutic effect, such compoundsare also named Active Pharmaceutical Ingredient (API).

According to another specific embodiment, said pharmaceuticalcomposition includes at least one further active ingredient in additionto said at least one glucocorticoids and said pharmaceuticallyacceptable liquid or solid carrier or carriers. The pharmaceuticalcomposition then includes one or more active agents together with one ormore of a variety of pharmaceutically acceptable carriers for deliveryto a subject, including a variety of diluents or excipients known tothose of ordinary skill in the art.

In a particular embodiment, said at least one further active ingredientis a non-antibiotic antimicrobial agent.

In animals especially, otitis externa is often linked to a parasiticinfestation, most often an otocariosis, or Otodectes cynotis (ear mites)infestation. Topical treatment with ear mites has often beenaccomplished with relatively long courses of topical insecticidaltherapy; e.g., with a pyrethrin-containing composition. However, shortercourses of therapy have been more recently obtained with mectin andmycin compounds; e.g., avermectins (such as ivermectin and selamectin)and milbemycin, administered otically, by injection, or on the skin. Ifclinically indicated, such anti-parasiticidal compounds may beco-administered within, or as a separate adjunct to, the compositions ofthe invention. Further, where clinically indicated, anti-viralcompounds, such as acyclovir, may be administered in lieu of, or as anadjunct to, antibiotic compounds.

Advantageously, pharmaceutical compositions according to the inventioncomprising a glucocorticoid and a further active ingredient as disclosedhere above enables to reduce the quantity of the said further activeingredients due to the mode of action of the glucocorticoid itself.According to the demonstration of the inventors, the glucocorticoid isby itself capable of treating infection and therefore, its combined useto typical active ingredients used in the treatment of epithelialmicrobial infections of fluid containing organ having a natural exteriororifice results in an improved treatment of the infection; quantities ofsaid other active ingredients can therefore be reduced.

The at least one glucocorticoid may also be administered together with amembrane restructuration protein such as glycoaminoglycan, ceramide,chitosan.

Another aspect of the invention is a composition comprising an activeingredient and a pharmaceutically acceptable carrier for the treatmentof an epithelial microbial infection of a fluid-containing organ havinga natural exterior orifice in a mammal, wherein said active ingredientis a glucocorticoid, preferably, hydrocortisone or a salt thereof, evenmore preferably hydrocortisone aceponate.

Another aspect of the invention is a pharmaceutical compositionconsisting of a glucocorticoid, preferably hydrocortisone or a saltthereof, even more preferably hydrocortisone aceponate, and apharmaceutically acceptable carrier for the treatment and/or theprevention of an epithelial microbial infection of a fluid-containingorgan having a natural exterior orifice, preferably the ear, in amammal, preferably a non-human mammal.

A particularly preferred pharmaceutically acceptable carrier is methylether propylene glycol.

The composition for administration may additionally include additives,excipients, thickeners, and other substances which allow for moreeffective administration. Examples include oils, emollients, or othersubstances which increase the effectiveness and comfort of ear drops,nasal sprays, and inhalable compositions. This may also includesubstances which enhance the smell or taste.

Preferred compositions and formulations according to the inventionprovides a global coverage of the organ having a natural exteriororifice to be treated locally.

Preferred excipients are those showing an antiseptic activity, they maybe selected in the group comprising, but not limited to alcohols (suchas Alcohol Benzoate, benzyl alcohol, ethanol), solvents (for examplePropylene glycol, Glycerine), surfactants, emollients (such as MacrogolCetostearyl Ether, Diisopropyl Adipate), dispersing agents (such asnikkol so-15 VL Sorbitan sesqui olate), flavoring agents (for examplevanillin), Chelating agents (for example EDTA disodium salt), oils (forexample olive oil (including Oleic Acid, linolenic and linoleic acids),coconut oil, Caprilic/Capric Triglyceride, Coco-Caprylate/Caprate),extracts (essentials oil or not) (such as turmeric oil, Melaleuca oil,calendula, oregon grape, thyme, garlic, sage, lemon grass, thija,peppermint, catnip, tea tree, hypericum, rosemary, citrus, eucalyptus,avies pectinata wood oils), additives (for example Nitric oxide, sodiumBenzoate, Silicon dioxide, Titanium dioxide, tanic acid, Curcumin(turmeric)), Polygodial, thymol, Nisine, polyhexanide, chlorobutanol,hydrogen peroxide, benzethonium chloride, silver (such as nitrate,chlorure or colloidal), silver salts, iodine or iodine derivated (suchas Povidone (PVP) iodine), disinfecting agents (for examplechlorhexidine, chloramine T, potassium permanganate), preservativeagents (for example Bronopol, E-280 (propionic acid), E281 (sodiumpropionate), E282 (calcium propionate), E36 (formic acid),Phenoxyethanol, Thimerosal, potassium sorbate, Polylysine, citric acid,mix of citric (25%), sorbic (16.7%) acids, sodium Thiosulfate, sodiumthiosulfate pentahydrate), sweetening agent (for example honey).

In a preferred embodiment, excipients having an antiseptic activity areselected in the group comprising Coco-Caprylate/Caprate, diisopropyladipate, coconut oil, Nisine, PVP iodine, Alcohol Benzoate,Caprilic/Capric Triglyceride, Glycerine, olive oil (including OleicAcid, linolenic and linoleic acids), Polylysine, Honey, Propyleneglycol, Macrogol Cetostearyl Ether, nikkol so-15 VL Sorbitansesquiolate, Silica dioxide.

In a more preferred embodiment, excipients having an antiseptic activityare PVP iodine, Coco-Caprylate/Caprate, diisopropyl adipate, coconut oiland Nisine.

A particularly preferred pharmaceutically acceptable carrier is methylether propylene glycol.

Other examples of formulations for topical application of glucocorticoidsuch as HCA particularly adapted for an otic administration include:

-   -   Suspension of lipid based particles with a solid matrix,        preferably Solid Lipid Nanoparticles (SLN) and Nanostructures        Lipid Carriers (NLC), comprising at least one lipid and        preferably at least one emulsifier. The lipid particles being        solid at room temperature, they can therefore comprise a single        solid lipid or a mixture of lipids that may partly contain at        least one solid lipid.

Suitable solid lipids are for example:

-   -   Hydrocarbons, such as solid paraffins;    -   Fatty acids, such as myristic acid, palmitic acid, stearic acid;    -   Monoglycerides, such as glyceryl monostearate, glyceryl        hydroxystearate, glyceryl benehate;    -   Diglycerides, such as glyceryl palmitostearate;    -   Triglycerides, such as glyceryl tristearate, glyceryl        trimyristate, hydrogenated castor oil, hydrogenated palm oil;    -   Waxes, such as beeswax, carnauba wax, cetyl palmitate.

A preferred solid lipid is glyceryl palmitostearate.

Suitable non solid lipids are for example:

-   -   Hydrocarbons, such as liquid paraffin, squalene (unsaturated        hydrocarbon);    -   Vegetable oils, such as soy bean oil, castor oil;    -   Fatty esters, such as isopropyl myristate;    -   Medium chain triglycerides, such as caprylic-capric        triglycerides;    -   Fatty acids, such as oleic acid, linoleic acid;    -   Propylene glycol fatty acid ester, such as propylene glycol        dicaprylocaprate;    -   Vitamin E.

The melting temperature range of solid lipids is preferably 30° C.-100°C. and more preferably 40° C.-90° C. When used in combination withnon-solid lipids, solid lipids can have a melting temperature that islower or higher than the 30° C.-100° C. range.

Generally, at least one surfactant is needed to aid in the dispersionprocess of the melted lipid(s) in the heated aqueous phase required toform a suspension of lipid based particles with a solid matrix and tostabilize the lipid particles dispersion after cooling.

Many class of surfactants, lipophilic or hydrophilic, can be used in thepreparation and stabilization of lipid particles suspension:

-   -   Non-ionic surfactants, such as polyoxyethylene sorbitan fatty        acid esters, sorbitan esters, macrogol 15 hydroxystearate,        glyceryl monostearate, polyoxyethylene castor oil derivatives,        polyoxylglycerides, glyceryl stearate and PEG-75 stearate, cetyl        alcohol and ceteth-20 and steareth-20;    -   Anionic surfactants, such as sodium dodecyl sulphate, sodium        deoxycholate, sodium glycocholate, sodium oleate;    -   Cationic surfactants, such as benzalkonium chloride, cetrimide,        stearylamine;    -   Amphoteric surfactants, such as egg lecithin, soya lecithin,        phosphatidylcholines, egg phospholipids, soya phospholipids,        phosphatidylethanolamines;    -   Block copolymers, such as poloxamers.

A combination of surfactants can be used, the choice of thesurfactant(s) depending on the lipid(s) used.

Preferably, non-ionic surfactants are used and more preferably thesurfactants are chosen from the polyoxyethylene sorbitan fatty acidesters group. A preferred surfactant is polysorbate 80.

Other excipients such as thickeners, emollients, co-surfactants,buffering agents, pH regulating agents, salts, antioxidants andpreservatives can be used for composition stability, more effectiveadministration and comfort of compositions.

A suspension of lipid based particles with a solid matrix can beprepared according to various standard methods. The preferred method ishigh-shear homogenization followed by ultrasonication. The lipid phasethat can contain at least surfactant(s) is melted. The glucocorticoid ispreferably dissolved or dispersed in the molten lipid. An aqueous phaseof the same temperature as the molten lipid phase and that can containat least surfactant(s) is added to the lipid phase. The API maynevertheless be in an aqueous phase. The mixture is then passed througha high-shear mixer to create a coarse emulsion, followed byultrasonication. The choice of the process parameters depends on theequipment and on the compositions.

-   -   In situ gelling systems that are liquid or viscous at room        temperature and that undergo gelation at body temperature,        comprising at least one polymer showing thermoreversible        gelation property, such as poloxamers.

A preferred polymer showing thermoreversible gelation property ispoloxamer 407.

Other excipients such as thickeners, solvents, emollients,co-surfactants, buffering agents, pH regulating agents, salts,antioxidants and preservatives can be used for composition stability,more effective administration and comfort of compositions.

An in situ gelling systems suspension can be prepared according tovarious methods. Preferably, all ingredients, except the poloxamer(s),are dissolved or dispersed in water. The poloxamer(s) is then dissolvedin the approximately 5° C. pre-cooled aqueous mixing. The activeingredient can be added alone or in combination with other excipient(s)in the aqueous phase containing the polymer such as poloxamer(s) at 5°C.

-   -   In situ phospholidpid gelling systems: said systems are fluid        and undergo gelation once they are in contact with water. This        is particularly adapted to otic administration after a clean up        of the ear before the treatment administration. Said gelling        systems enable a better residency of the product locally. Such        systems may comprises at least one phospholipid, such as        phosphatidylcholine derivatives.

A preferred jellifying agent is Phospholipon 90G.

Other excipients such as thickeners, solvents, emollients,co-surfactants, buffering agents, pH regulating agents, salts,antioxidants and preservatives can be used for composition stability,more effective administration and comfort of compositions.

Examples of preferred solvents include isopropyl myristate and ethanol.

-   -   Jellified formulations, which present improved viscosity and        therefore enables an improved residency of the product in the        ear. Such formulations may comprise thickeners. Examples of        thickeners comprises Povidone K90, Glycerol, PEG400, Carbomers        (Acrypol® 971)/acrylic polymers, Hypromellose (Hydroxypropyl        Methylcellulose 2910 60HD6), Hydroxypropyl methyl cellulose and        other common thickeners (gums, poloxamers). Preferred thickeners        are Povidone K90 and glycerol.

Other excipients such as solvents, emollients, co-surfactants, bufferingagents, pH regulating agents, salts, antioxidants and preservatives canbe used for composition stability, more effective administration andcomfort of compositions.

-   -   Micellar solutions, comprising at least one dispersed        surfactant. At least one non solid lipid can be added.

Suitable surfactants are preferably hydrophilic, exhibiting a highhydrophilic-lipophilic balance value, being soluble in water and actingas solubilizing agents for lipophilic substances by keeping these inmicellar solution. More preferably, surfactants are non-ionic such aspolyoxyethylene sorbitan fatty acid esters, polyoxyethylene castor oilderivatives, macrogol 15 hydroxystearate, polyoxylglycerides andpoloxamers.

A preferred surfactant is polysorbate 80.

Other excipients such as thickeners, solvents, emollients,co-surfactants, buffering agents, pH regulating agents, salts,antioxidants and preservatives can be used for composition stability,more effective administration and comfort of compositions.

Micellar solutions are prepared by adding sufficient surfactantconcentration in a liquid medium to form the micellar solutionSurfactant(s) concentration should be above the critical micellarconcentration to predominantly form micelles. The lipophilicsubstance(s) is then added and solubilized by the micellar solutionunder gentle agitation.

-   -   Cyclodextrin inclusion complexes, comprising at least one        cyclodextrin acting as solubilizing agents for lipophilic        substances by keeping these entrapped in the hydrophobic        internal cavity of the solubilized cyclodextrin molecules.

Other excipients such as thickeners, solvents, emollients,co-surfactants, buffering agents, pH regulating agents, salts,antioxidants and preservatives can be used for composition stability,more effective administration and comfort of compositions.

A preferred cyclodextrin is hydroxypropyl-beta-cyclodextrin.

Aqueous cyclodextrin inclusion complexes are prepared by addingcyclodextrin(s) in aqueous medium. The lipophilic substance(s) is thenadded and solubilized by the cyclodextrin(s) molecules under gentleagitation.

Other excipients such as thickeners, solvents, emollients,co-surfactants, buffering agents, pH regulating agents, salts,antioxidants and preservatives can be used for composition stability,more effective administration and comfort of compositions.

-   -   Oil/Water or water/oil Emulsions: emulsions comprising at least        one lipid, at least one emulsifier and at least water.

Suitable lipids are for example:

-   -   Hydrocarbons, such as solid paraffins, liquid paraffin, squalene        (unsaturated hydrocarbon);    -   Fatty acids, such as myristic acid, palmitic acid, stearic acid;    -   Monoglycerides, such as glyceryl monostearate, glyceryl        hydroxystearate, glyceryl benehate;    -   Diglycerides, such as glyceryl palmitostearate;    -   Triglycerides, such as glyceryl tristearate, glyceryl        trimyristate, hydrogenated castor oil, hydrogenated palm oil,        soy bean oil, castor oil, caprylic-capric triglycerides;    -   Waxes, such as beeswax, carnauba wax, cetyl palmitate;    -   Fatty esters, such as isopropyl myristate;    -   Fatty acids, such as oleic acid, linoleic acid;    -   Propylene glycol fatty acid ester, such as propylene glycol        dicaprylocaprate;    -   Vitamin E.

Generally, at least one surfactant is needed to aid in the dispersionprocess and to stabilize the emulsion. A combination of surfactants canbe used.

Many class of surfactants, lipophilic or hydrophilic, can be used:

-   -   Non-ionic surfactants, such as polyoxyethylene sorbitan fatty        acid esters, sorbitan esters, macrogol 15 hydroxystearate,        glyceryl monostearate, polyoxyethylene castor oil derivatives,        polyoxylglycerides, glyceryl stearate and PEG-75 stearate, cetyl        alcohol and ceteth-20 and steareth-20;    -   Anionic surfactants, such as sodium dodecyl sulphate, sodium        deoxycholate, sodium glycocholate, sodium oleate;    -   Cationic surfactants, such as benzalkonium chloride, cetrimide,        stearylamine;    -   Amphoteric surfactants, such as egg lecithin, soya lecithin,        phosphatidylcholines, egg phospholipids, soya phospholipids,        phosphatidylethanolamines;    -   Block copolymers, such as poloxamers.

Preferably, non-ionic surfactants are used.

Other excipients such as thickeners, emollients, co-surfactants,buffering agents, pH regulating agents, salts, antioxidants andpreservatives can be used for composition stability, more effectiveadministration and comfort of compositions.

Formulations particularly adapted to treat mastitis for non-humananimals, also called intramammary infusion, are usually composed of avehicle or carrier associated with an active ingredient. Intramammaryformula can be in the form of a liquid product, a semi-solid or athixotropic product such as a solution, a suspension, a paste, a gel, ora cream formulation.

In a preferred embodiment the composition according to the inventioncomprises at least one glucocorticoid, as described above, which issolubilized in the pharmaceutically acceptable carrier. In anotherpreferred embodiment, the glucocorticoid according to the invention isin the form of particles in the intramammary formulations.

The intramammary composition of the invention may further comprise anysuitable pharmaceutically acceptable ingredients which are known in thepharmaceutical art, selected from the groups consisting of: oil, mediumchain triglyceride, wax (e.g. microcrystalline), fatty acid, fatty acidderivatives, viscosity control agent or thickener (stearate salt,silicon dioxide, cellulose); pigment, opacifier, dispersing agent;emulsifier; stabilizer; surfactant, humectant; antioxidant;antibacterial agent, antifungal agent, preservative, emollient,polymers, any compound conferring organoleptic properties (flavor, dye,perfume, etc.), water, and combinations thereof. The oil used isselected from vegetable (e.g. natural plant oil) or mineral origin (e.g.paraffin, white petrolatum, yellow petrolatum, etc.). The fatty acidderivatives are either organic or inorganic compounds such as aluminiumstearate, magnesium stearate, zinc stearate, stearic acid, hydrogenatedvegetable oil and the like. Mineral particles such as, but not only,silica, clay, calcium carbonate are eventual excipients that can be usedas thickener. The formula can also comprise a non-toxic heavy metal.

An intramammary infusion comprising a glucocorticoid according to theinvention in the form of particles which may be encapsulated in thepharmaceutically acceptable carrier targets a protection with a longlasting effect, to ensure the desired treatment time and efficacy.

Compositions according to the invention can be administered throughvarious administration routes, including but not limited to topical, ororal administrations, especially when the target is digestive tract andintestinal epithelium.

Preferably, compositions according to the invention are administered bytopical application or topical administration onto an epithelial area ofthe fluid-containing organ having a natural exterior orifice.Particularly, said compositions are administered by topical applicationor topical administration into, i.e. inside the cavity of, the fluidcontaining organ having a natural exterior orifice, i.e. through theorifice of said fluid-containing organ and onto an epithelial area ofsaid fluid-containing organ affected by the microbial disorder.

Preferably, the present invention also provides methods for topicallytreating epithelial microbial infections through topical administrationof glucocorticoids on said epithelia of a fluid containing organs havinga natural exterior orifice, particularly into, i.e. inside the cavityof, the fluid containing organ having a natural exterior orifice, i.e.through the orifice of said fluid-containing organ and onto anepithelial area of said fluid-containing organ affected by the microbialdisorder.

By “topical administration” is meant that the at least oneglucocorticoid used according to the invention is applied onto theepithelial area that is (treatment) or that has been (prevention ofrecurrence) affected by the microbial disorder.

In a particular embodiment, the at least one glucocorticoid, preferablyhydrocortisone or a salt thereof, even more preferably hydrocortisoneaceponate, used according to the invention is applied to the externalear canal; i.e., on the outer ear side of the tympanic membrane(eardrum). Topical administration to the outer ear canal is achievedvia, for example, introducing the composition of the invention into theouter ear canal via any medically acceptable means; e.g., by applyingthe carrier composition to the membrane by insertion of a needlelesssyringe, dropper or swab into the auditory canal. Administration isrepeated as required to achieve the therapeutically effective dosagelevel for the glucocorticoid compound given.

Where the method of the invention comprises injection or infusion of theat least one glucocorticoid into an udder via the teat canal or into theuterus via the vagina, it can provide effective treatment of mastitisand metritis/endometritis, respectively. “Intramammary infusion” is anoperation wherein a liquid composition is caused to flow into an udder(into the teat cistern and/or the gland cistern) via a teat canal,regardless of the timescale involved. “Uterus infusion” is an operationwherein a liquid composition is caused to flow into the uterus via thevagina, regardless of the timescale involved. In the present context,“infusion” and “injection” are substantially synonymous.

In a particular embodiment, the at least one glucocorticoid can beadministered for treatment of mastitis by inserting the cannula nozzleof a mastitis syringe into the external orifice of the teat canal of anudder of a milk producing animal and infusing the at least oneglucocorticoid into the udder.

In a particular embodiment, the at least one glucocorticoid can beadministered for treatment of metritis and/or endometritis by insertingthe cannula nozzle of a syringe into the vagina of the mammal having auterus and infusing the at least one glucocorticoid into the vaginaand/or the uterus of said animal.

For the treatment and the prevention of epithelial microbial infectionsof a fluid containing organs having a natural exterior orificeencompassed by the present invention, a quantity of 0.01 to 150 mg ofglucocorticoid is administered at each administration. Accordingly, thepharmaceutical composition of the invention comprises a content ofglucocorticoid chosen so that a single dose of said pharmaceuticalcomposition comprises the required quantity of glucocorticoid.

Such glucocorticoid may be administered once or twice a day for 1 to 20days; depending on the infection importance, the glucocorticoid may beadministered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 or 20 days (i.e. up to 20 days).

In the specific case of the treatment of otitis externa, the quantity ofglucocorticoid, preferably hydrocortisone or a salt thereof, even morepreferably hydrocortisone aceponate, to be applied is preferablycomprised between 0,01 and 10 mg; such glucocorticoid may beadministered once or twice a day for 2 to 20 days; depending on theinfection importance, the glucocorticoid may be administered for 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.

Preferably, for the treatment of otitis externa the quantity ofglucocortoid, preferably hydrocortisone or a salt thereof, even morepreferably hydrocortisone aceponate, to be administered is comprisedbetween 0,05 and 5 mg, even more preferably between 0,1 and 1.2 mg. Evenmore preferably, the quantity of glucocorticoid is comprised between 0,1to 0.5 mg. In a most preferred embodiment, said quantity is comprisedbetween 0,25 to 0.3 mg.

For example, 0.5 ml of a pharmaceutical composition according to theinvention is used and in which the content of glucocorticoid in saidpharmaceutical composition is 0,584 mg/ml.

In another preferred embodiment, said glucocorticoid, preferablyhydrocortisone or a salt thereof, even more preferably hydrocortisoneaceponate, for use in the treatment of otitis externa is applied once ortwice daily from 1 to 14 days, and preferably from 7 to 14 days, in theear. In a most preferred embodiment, said glucocorticoid for use in thetreatment of externa otitis is administered once daily for 7 to 14 days.

According to a preferred embodiment related to the treatment of externaotitis, the quantity of glucocorticoid, preferably hydrocortisone or asalt thereof, even more preferably hydrocortisone aceponate, to beadministered is comprised between 0,1 to 0.5 mg and is administered oncedaily for 7 to 14 days, preferably for 7 days.

According to a more preferred embodiment related to the treatment ofexterna otitis, said pharmaceutical composition comprises HCA.Preferably the animal is a dog. Preferably said dog is not an atopicdog. Preferably, said dog is a cocker.

According to another embodiment, the treatment may be administeredsequentially. The term “sequentially” or “sequential treatment”corresponds to an administration of the glucocorticoid over a firstperiod of treatment, followed by a second period where the treatment isstopped, followed by an additional period of treatment. The sequentialtreatment may be repeated as long as necessary.

According to another embodiment related to the prevention of externaotitis, the quantity of glucocorticoid, preferably hydrocortisone or asalt thereof, even more preferably hydrocortisone aceponate, to beadministered is comprised between 0,01 and 10 mg and is administered atleast once a week in the ear for 10 to 20 weeks.

Preferably, for the prevention of otitis externa, the quantity ofglucocortoid, preferably hydrocortisone or a salt thereof, even morepreferably hydrocortisone aceponate, to be administered is comprisedbetween 0,05 and 5 mg, even more preferably between 0,1 and 1.2 mg. Evenmore preferably, the quantity of glucocorticoid is comprised between 0,1to 0.5 mg. In a most preferred embodiment, said quantity is 0,25 to 0.3mg.

Preferably said glucocorticoid is HCA. Preferably the animal is a dog.Preferably said dog is not an atopic dog. Preferably, said dog is acocker.

In another preferred embodiment, said pharmaceutical compositioncomprising a glucocorticoid, preferably hydrocortisone or a saltthereof, even more preferably hydrocortisone aceponate, for use in theprevention of otitis externa is applied in the ear at least once a week,preferably twice a week for 10 to 20 weeks, preferably 12 to 18 weeks,even more preferably for 16 weeks. Preferably, the composition isadministered on two consecutive days per week.

According to a preferred embodiment related to the treatment of externaotitis, of the quantity of glucocorticoid, preferably hydrocortisone ora salt thereof, even more preferably hydrocortisone aceponate, to beadministered is comprised between 0,1 to 0.5 mg and is administered inthe ear twice a week for 16 weeks.

Preferably said glucocorticoid is HCA. Preferably the animal is a dog.Preferably said dog is not an atopic dog. Preferably, said dog is acocker.

In the specific case of the treatment of mastitis, the quantity ofglucocorticoid to be applied in the udder via the teat canal ispreferably comprised between 1 and 50 mg; such glucocorticoid may beadministered at least once a day for 1 to 10 days; depending on theinfection importance, the glucocorticoid may be administered for 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 days.

Preferably, the quantity of glucocorticoid to be administered iscomprised between 10 and 40 mg, even more preferably between 15 and 25mg. A most preferred embodiment is a dose of glucocorticoid of 20 mg.

For example, a volume of 10 ml of a pharmaceutical compositioncomprising the glucocorticoid according to the invention is administeredin the udder via the teat canal and the concentration of saidglucocorticoid in said pharmaceutical composition is preferablycomprised between 0,05 and 1% w/w, preferably 0,2%.

Preferably, the pharmaceutical composition for the treatment of mastitiscomprising a glucocorticoid according to the invention is administeredin the udder via the teat canal once or twice per day, preferably twiceper day, for 1 to 10 days, preferably for 1 to 5 days, and even morepreferably for 1 up to 2 days.

In a most preferred embodiment, the glucocorticoid according to theinvention is administered in the udder via the teat canal with aquantity of glucocorticoid comprised between 10 and 40 mg twice per dayfor up to 2 days.

In a preferred embodiment, each teat of the udder of the mammal istreated with a pharmaceutical composition according to the invention.Preferably said glucocorticoid is HCA. Preferably the animal is a cow.

According to another embodiment related to the prevention of mastitis,the quantity of glucocorticoid to be administered is comprised between0,01 and 50 mg, preferably between 10 and 40 mg, even more preferablybetween 15 and 25 mg and is administered in the udder via the teat canalat least once a week. Preferably said glucocorticoid is HCA. Preferablythe animal is a cow.

In the specific case of the treatment of metritis/endometritis, thequantity of glucocorticoid to be applied in the uterus is preferablycomprised between 1 and 150 mg, preferably between 10 and 120 mg, evenmore preferably between 30 and 120 mg.

According to one embodiment related to the treatment ofmetritis/endometritis, said glucocorticoid may be administered once orseveral times. In a preferred embodiment, said glucocorticoid isadministered once. Preferably said glucocorticoid is HCA.

According to another embodiment related to the prevention ofmetritis/endometritis, the quantity of glucocorticoid is comprisedbetween 1 and 150 mg, preferably between 10 and 120 mg, even morepreferably between 30 and 120 mg, and is administered in the uterus onceor several times.

The present invention further provides a method of prevention ortreatment of an epithelial microbial infection of a fluid containingorgans having a natural exterior orifice, preferably the ear, the methodcomprising administering topically at least one glucocorticoid,preferably hydrocortisone or a salt thereof, even more preferablyhydrocortisone aceponate, to said epithelium affected by said microbialinfection.

Another aspect of the invention is a kit comprising:

-   -   a) a pharmaceutical composition according to the invention,    -   b) a notice of use of the product, and    -   c) an optional administration device.

Said pharmaceutical composition in the kit according to the inventionmay be provided in a ready-to-use administration device or may beprovided separately from the administration device.

Examples of administration device include, but are not limited to, pumpspray, syringe, tube.

A particularly adapted administration device for the treatment and/orprevention of otitis externa is a pump with a canula.

A particularly adapted administration device for the treatment and/orprevention of mastitis, metritis and/or endometritis is a syringe.

EXAMPLE 1 Evaluation of the Efficacy of Hydrocortisone Aceponate (HCA)Formulation (Cortavance®) for the Treatment of Otitis Externa in Dogs

Objectives: Erythroceruminous otitis is mainly due to inflammation whilemicrobial proliferation being only a secondary manifestation of thedisease. The efficacy of local corticotherapy with Hydrocortisoneaceponate (HCA) as a stand-alone therapy has been previously evaluatedfor the prevention of relapse of allergic otitis, but there is noconvincing data in veterinary medicine about its use for the treatmentof an acute flare of otitis externa. The main objective of this studywas to evaluate if the use of HCA alone in the ear canal can beeffective in the treatment of otitis externa in dogs.

Cortavance® product is composed of 0,0584% of Hydrocortisone aceponatein Propylene glycol methyl ether (QSP 100%).

Materials and methods: Dogs with clinical (OTIS3 score≥4) andcytological (score>1) signs of acute episode of otitis externa(erythroceruminous, non-parasitic), were included in thisdouble-blinded, randomized, controlled, multicenter clinical fieldtrial. Dogs were allocated in two groups and the ear canal was treatedwith 0.5 mL of HCA (Cortavance® group) once daily or 0.5 mL of apharmaceutical veterinary treatment Surolan® comprising a combination ofan antifungal, anti-inflammatory and antibiotic agents (miconazole,prednisolone and polymyxine B) (Surolan® group) twice daily, for 7 daysor 14 days if symptoms persisted. At D7, D14 and D28, clinical OTIS3score (according to Nuttal and Bensignor, Vet dermatol, 2014),cytological score (according to Budach and Mueller, Vet Derm, 2012) andpruritus (according to Rybnicek and al, Vet Derm, 2008) were compared tobaseline. The treatment was considered as a success if remissionoccurred on D7 or D14 with no relapse at D28.

Results: Fifty three dogs with unilateral or bilateral otitis externawere enrolled in this clinical field trial, but 4 dogs were excludedfrom the analysis because of non-compliance to the protocol (dogs onlyseen the first or the first two follow-up visits). The analysis wasperformed on 49 dogs (73 ears): 23 dogs (34 ears) and 26 dogs (39 ears)were allocated in Cortavance® group and Surolan® group respectively.

At baseline, homogeneity was respected between Cortavance® vs Surolan®groups respectively for body weight (21.42 kg±13.61 vs 19.05 kg±16.11,Wilcoxon's test p=0.37), otitis unilateral or bilateral (52.2% vs 50.0%unilateral or 47.8% vs 50.0% bilateral, Chi-square test p=0.88),clinical score OTIS3 (6.2±0.9 vs 6.0±1.4, Wilcoxon's test p=0.35),global cytological score (4.0±1.6 vs 4.5±1.7, Wilcoxon's test p=0.24),type of otitis (bacteria: 5.9% vs 5.1%, bacteria and Malassezia: 61.8%vs 74.4%, Malassezia: 32.3% vs 20.5%, Fisher's exact test p=0.47) andpruritus score (5.9±1.8 vs 5.7±2.2, Student's test p=0.72). The dogswere significantly younger (4.24 years±3.26 vs 6.16 years±3.30,Wilcoxon's test p=0.02) for the group Cortavance® vs group Surolan®respectively. At D7, both treatment improved clinical, cytological andpruritus signs with a success rate not significantly higher (50.0% vs30.8%, Chi-square's test p=0.09) for Cortavance® vs Surolan®respectively. While global cytological score reduction were similarbetween groups (61.5%±23.4 vs 61.7%±22.7, Wilcoxon's test p=0.89),clinical score reduction (62.7%±22.6 vs 46.3%±20.8, Student's testp=0.002) and pruritus reduction (70.0%±17.5 vs 57.1%±25.3, Student'stest p=0.047) were significantly higher for Cortavance® vs Surolan®respectively.

At D14, the success rate was similar (74.1% vs 73.5%, Chi-square's testp=0.96) and clinical score reduction (82.3%±15.4 vs 77.6%±18.5,Wilcoxon's test p=0.27), global cytological score reduction (82.2%±26.7vs 76.2%±28.3, Wilcoxon's test p=0.31) and pruritus reduction (88.9%±9.8vs 80.5%±19.2, Wilcoxon's test p=0.15) were not significantly higher forCortavance® vs Surolan® respectively.

At D28, the no relapse rate was not significantly lower (83.3% vs 95.2%,Fisher's exact test p=0.35) and clinical score reduction (80.2%±24.3 vs71.1%±22.7, Wilcoxon's test p=0.07), cytological score reduction(84.1%±19.7 vs 73.5%±26.1, Wilcoxon's test p=0.09) and pruritusreduction (86.1%±20.0 vs 76.3%±21.4, Wilcoxon's test p=0.08) were notsignificantly higher for Cortavance® vs Surolan® respectively.

A complementary analysis was performed by considering individually thecytological scores for Bacteria, Malassezia and Neutrophils. Score forBacteria compared to baseline was reduced by 58.7%±35.1 vs 49.7%±34.5(Wilcoxon's test p=0.45) at D7, 84.4%±30.1 vs 69.9%±39.2 (Wilcoxon'stest p=0.22) at D14 and 80.3%±22.8 vs 70.8%±34.4 (Wilcoxon's testp=0.44) at D28 for Cortavance® vs Surolan® respectively. Score forMalassezia compared to baseline was reduced by 58.6%±31.9 vs 63.1%±26.8(Wilcoxon's test p=0.64) at D7, 79.2%±31.4 vs 77.8%±41.3 (Wilcoxon'stest p=0.87) at D14 and 84.1%±23.6 vs 72.8%±37.5 (Wilcoxon's testp=0.28) at D28 for Cortavance® vs Surolan® respectively. Note that onlyone ear had an increase in Malassezia score during the course of thestudy and that this ear was in Surolan® group. Score for Neutrophilscompared to baseline was reduced by 75.0%±46.3 vs 58.3%±46.9 (Wilcoxon'stest p=0.41) at D7, 100%±0.0 vs 81.8%±40.5 (Wilcoxon's test p=0.28) atD14 and 100% vs 100% at D28 for Cortavance® vs Surolan® respectively.

The global success rate was not significantly higher (60.6% vs 57.1%,Chi-square's test p=0.77) for Cortavance® compared to Surolan®respectively. Moreover statistical analysis further emphasis the betteronset of action of Cortavance® from D7, where both pruritus and clinicalscore are significantly lower compared to Surolan®.

No adverse events were recorded during the trial.

Conclusion: This clinical field study highlights that HCA is at least aseffective as a reference treatment comprising anti-inflammatory,antibiotic and antifungal agents in treating erythemato ceruminousotitis externa in dogs, and to control bacterial and fungalproliferations.

EXAMPLE 2 Evaluation of the Efficacy of Hydrocortisone Aceponate (HCA)Formulation for the Intramammary Treatment of Mastitis in Lactating Cowsand Heifers

The purpose of this multicenter clinical field study is to evaluate theefficacy and the safety of HCA at a dosage of 20 mg in 10 ml of Vaselineoil based formulation every 12±2 h hours (minimum of one and maximum offour treatments) in the intramammary treatment of mild clinical mastitisin lactating cows; more specifically to assess the efficacy (clinicalcure rate) of HCA alone in the treatment of udder inflammation (mildclinical mastitis), under field conditions and to assess the safety andthe risk of aggravation or relapses during and after the treatment withHCA alone.

Animals (50 lactating cows and heifers presenting clinical symptoms ofmastitis) have been intramammarily treated with one syringe (10 mL)containing 20 mg of HCA at each time point for a minimum of 1 and amaximum of 4 treatments in a 12±2 h interval (each morning or eveningmilking time, respectively). Depending on the result of the clinicalexamination and the clinical status classification, the animal have beentreated until the animal was classified as clinically cured, failure orthe maximum of 4 treatments is reached. Before the tested treatment, asample of milk of each animal has been collected to be tested forbacteriology of classical pathogenic bacteria; among the 50 animals, 34produced a milk containing pathogenic bacteria, no pathogenic bacteriahas been detected in the milk of the 16 remaining animals, it cannevertheless not be excluded that those 16 animals were infected by anon-tested pathogenic bacteria.

Results: No adverse effect of the treatment has been observed, it hasbeen well tolerated. 16/50 animals have been cured (32%) after 3 or 4administrations of HCA; among those animals, 7/34 animals which werebacteriologically positive at DO (assessed in milk sample collected forbacteriology before the treatment starts) were cured (20.6%) and 9/16animals which were bacteriologically negative at DO were cured (56.3%).

Conclusion: This clinical field study highlights that HCA is able totreat infectious mastitis; it could thus be used as a first-linetreatment to limit as much as possible the use of antibiotics and theonset of antimicrobial resistance.

EXAMPLE 3 Solid Lipid Nanoparticles Formula

Name of substances Quantity percentage (% w/w) Hydrocortisone aceponate0.058 Precirol ATO 5 10.0 Polysorbate 80 2.5 Purified water buffered atQSP 100% pH 4.75 (acetate buffer)

EXAMPLE 4 In Situ Gelling System

Name of substances Quantity percentage (% w/w) Hydrocortisone aceponate0.058 Xanthan gum 0.75 Kolliphor P407 17.0 Purified water buffered atQSP 100% pH 4.75 (acetate buffer)

EXAMPLE 5 Micellar Solution

Name of substances Quantity percentage (% w/w) Hydrocortisone aceponate0.1 Polysorbate 80 5.0 Purified water buffered at QSP 100% pH 4.75(acetate buffer)

EXAMPLE 6 Cyclodextrin Inclusion Complex Formulation

Name of substances Quantity percentage (% w/w) Hydrocortisone aceponate0.1 Hydroxypropyl-beta- 10.0 cyclodextrin Purified water buffered at QSP100% pH 4 (acetate buffer)

EXAMPLES 7 Emulsions

Emulsion 1

Name of substances Quantity percentage (% w/w) Hydrocortisone aceponate0.13 Emulcire 61 WL 2659 4.0 Apifil 7.0 Miglyol 812N 29.87 Purifiedwater buffered at QSP 100% pH 4.75 (acetate buffer)

Emulsion 2

Name of substances Quantity percentage (% w/w) Hydrocortisone aceponate0.13 Emulcire 61 WL 2659 3.0 Gelot 64 3.0 Cetyl alcohol 3.0 Miglyol 812N14.87 Purified water buffered at QSP 100% pH 4.75 (acetate buffer)

Emulsion 3

Name of substances Quantity percentage (% w/w) Hydrocortisone aceponate0.13 Emulcire 61 WL 2659 3.0 Apifil 5.0 Miglyol 812N 29.87 Purifiedwater buffered at QSP 100% pH 4.75 (acetate buffer)

EXAMPLE 8 Intramammary Formulations

% w/w A B C D E F Hydrocortisone  0.22  0.23  0.21  0.21  0.4  0.46aceponate Vaseline oil 68.6 40 / / 40 40 Miglyol 812N / QS100 QS100 / /Vaseline Codex T5 / 1 / / QS100 QS100 Vaseline 7702 28.18 QS100 /  1 / /Aluminium  3 / 10  5 / / monostearate Thixin R / / / / / % w/w G H I J KL Hydrocortisone /  0.23  0.23  0.23  0.23  0.23 aceponate Vaseline oilQS 35 40 45 50 55 Miglyol 812N / / / / / / Vaseline Codex  1 QS100 QS100QS100 QS100 QS100 T5 Vaseline 7702 / / / / / / Aluminium / /  1 /  1  1monostearate Thixin R  4 / / / /

EXAMPLE 9 Intramammary Formulations Associated with Antibiotics

% w/w M N 0 P Q R S T Hydrocortisone  0.23  0.23  0.23  0.23 0.23 0.230.23 0.23 aceponate Cephalexin H2O  2.49  2.49  2.49  2.49 2.49 2.492.49 2.49 microsterile Sterile Kanamycine  1.88  1.88  1.88  1.88 1.881.88 1.88 1.88 monosulfate Vaseline oil 60 60 60 70 QS100 / / QS100Liquid paraffin / / / / / QS100 / 1 Miglyol 812N  1 / / / / QS100 /Vaseline Codex T5 QS100 QS100 / / / / / / Vaseline 7702 / / QS100 QS1001 1 1 1 Aluminium /  3 /  3 7.5 7.5 7.5 / monostearate Thixin R / / /  1/ / / 3.5

EXAMPLE 10 In Situ Gelling Phospholipid Systems for Otic Administration

Name of substances A (% w/w) B (% w/w) C (% w/w) D (% w/w) E (% w/w)Hydrocortisone Active  0.0584  0.0584  0.0584  0.0584  0.0584 aceponatePhospholipon 90G Jellifying agent 70 50 70 36 25 Isopropyl myristateSolvent — QSP100 15 54 QSP100 Ethanol Solvent QSP100 — QSP100 QSP100 —

EXAMPLE 11 Jellified Formulations for Otic Administration

System 1

Name of substances Quantity percentage (% w/V) Hydrocortisone aceponate0.0584 PVPK90 2 Dowanol QS 100

Said formulation provided an improved viscosity by 7 compared toCortavance®

System 2

Name of substances Quantity percentage (% w/V) Hydrocortisone aceponate0.0584 PVPK90 1 Glycerol 10 Dowanol QS 100

Said formulation provided an improved viscosity by 12 compared to theCortavance® formulation.

Such formulations with an increased viscosity enable to increase theresidence time of the product in the ear, hence increase the duration ofactivity of the product. This presents the advantage of enabling reducednumber of administration.

Said systems 1 and 2 were tested for in vivo tolerance in dogs. Theformulations were administered at a dose of 1 ml once a day during 3consecutive days in dog ear and were well tolerated.

1. A method for the treatment and/or the prevention of an epithelialmicrobial infection of the udder in a non-human mammal in need thereof,the method comprising topically administering in the udder to be treatedan effective amount of hydrocortisone, a monoester, a diester or a saltthereof, wherein said epithelial microbial infection of the udder ismastitis.
 2. The method of claim 1, wherein said epithelial microbialinfection of the udder is caused by bacteria, fungi and/or yeasts. 3.The method of claim 2, wherein said epithelial microbial infection ofthe udder is caused by bacteria.
 4. The method of claim 2, wherein thebacteria is selected in the group comprising Staphylococcus spp.,Escherichia coli, Klebsiella spp., Trueperella pyogenes, Streptococcusspp., Corynebacterium spp., Enterococcus spp., Pseudomonas spp.,Mycoplasma spp., Prototheca spp., Candida spp., Rhodotorula,Trichosporon, Saccharomyces, Pichia, Cryptococcus, Aspergillus,Penicillum, Epicoccum, Phoma and Alternaria.
 5. The method of claim 2,wherein said epithelial microbial infection of the udder is caused byfungi and/or yeasts.
 6. The method of claim 1, wherein said non-humanmammal is a non-human lactating mammal.
 7. The method of claim 1 for thetreatment of mastitis, wherein the quantity of hydrocortisone,monoester, diester or salt thereof to be applied in the udder iscomprised between 1 and 50 mg and is administered at least once a dayfor 1 to 10 days.
 8. The method of claim 1 for the prevention ofmastitis, wherein the quantity of hydrocortisone, monoester, diester orsalt thereof to be applied in the udder is comprised between 1 and 50 mgand is administered at least once a week.
 9. The method of claim 1,wherein said hydrocortisone, monoester, diester or salt thereof isformulated in a pharmaceutical composition also comprising apharmaceutically acceptable carrier, and wherein said composition doesnot contain any antibiotic agent, antifungal agent and/or antiviralagent.
 10. The method of claim 1, wherein said composition isadministered by topical application into the udder via the treat canal.